We have created a nonmuscle myosin heavy chain II-A (NMHC II-A) null mouse in order to study the function of this myosin II isoform and its relation to the other nonmuscle myosin isoforms in its class (NMHC II-B and -C). Exon 3, near the ATP binding region of the myosin heavy chain, was targeted by insertion of the neomycin cassette. In contrast to the NMHC II-B null mouse, which dies around E12 to birth with severe defects in the heart and brain, the deletion of NMHC II-A results in lethality at E7.5 or earlier. NMHC II-A null embryos are smaller than wild type or heterozygous littermates and are severely disorganized. Embryonic stem (ES) cells express NMHC II-A and II-B, but not II-C. A-/A- ES cells were generated by re-electroporation of A+/A- cells followed by selection at increased concentrations of G418. Because of the early lethality of the NMHC II-A null mice, embryoid bodies which recapitulate the early stages of embryonic development were formed from these cells and studied along with A+/A- and wild type embryoid bodies. Conventional RT-PCR and real time RT-PCR showed that some markers of visceral endoderm specification are present in all three types of embryoid bodies. However, a number of markers of visceral endoderm and mesoderm development are missing or reduced in the null embryoid bodies. Attempts to rescue A-/A- ES cell by introduction of GFP tagged NMHC II-A have, to date, been unsuccessful. Additionally, in an attempt to study myosin II-A function in the adult, mice have been created which contain the neomycin cassette in an intron, possibly creating a hypomorph which expresses low levels of NMHC II-A. The presence of loxP sites flanking the neomycin cassette and the nearby exon 3 will also permit ablation of the exon in a time-dependent manner. This is accomplished by crossing the mice which are homozygous for the targeted allele to mice expressing Cre recombinase and rtTA, the transactivator of the recombinase. And finally, we have created a mouse bearing a mutation in NMHC II-A which compromises myosin activity and mimics a mutation found in certain human disease states.